Preclinical evaluation of the PARP inhibitor BMN-673 for the treatment of ovarian clear cell cancer.

PURPOSE: To determine if models of ovarian clear cell carcinomas (OCCCs) harbouring defects in homologous recombination (HR) DNA repair of double strand breaks (DSBs) are sensitive to cisplatin and/or PARP inhibition. EXPERIMENTAL DESIGN: The HR status of 12 OCCC cell lines was determined using RAD51/γH2AX foci formation assays. Sensitivity to cisplatin and the PARP inhibitor BMN-673 was correlated with HR status. BRCA1, BRCA2, MRE11 and PTEN loss of expression was investigated as a potential determinant of BMN-673 sensitivity. A tissue microarray containing 50 consecutive primary OCCC was assessed for PTEN expression using immunohistochemistry. RESULTS: A subset of OCCC cells displayed reduced RAD51 foci formation in the presence of DNA DSBs, suggestive of HR defects. HR-defective OCCC cells, with the exception of KOC-7c, had higher sensitivity to cisplatin/ BMN-673 than HR-competent OCCC cell lines (Log10 SF50 -9.4 (SD +/- 0.29) vs -8.1 (SD +/- 0.35), mean difference 1.3, p < 0.01). Of the cell lines studied, two, TOV-21G and KOC-7c, showed loss of PTEN expression. In primary OCCCs, loss of PTEN expression was observed in 10% (5/49) of cases. CONCLUSIONS: A subset of OCCC cells are sensitive to PARP inhibition in vitro, which can be predicted by HR defects as defined by γH2AX/RAD51 foci formation. These results provide a rationale for the testing of HR deficiency and PARP inhibitors as a targeted therapy in a subset of OCCCs.

Intra-tumor genetic heterogeneity and alternative driver genetic alterations in breast cancers with heterogeneous HER2 gene amplification.

BACKGROUND: HER2 is overexpressed and amplified in approximately 15% of invasive breast cancers, and is the molecular target and predictive marker of response to anti-HER2 agents. In a subset of these cases, heterogeneous distribution of HER2 gene amplification can be found, which creates clinically challenging scenarios. Currently, breast cancers with HER2 amplification/overexpression in just over 10% of cancer cells are considered HER2-positive for clinical purposes; however, it is unclear as to whether the HER2-negative components of such tumors would be driven by distinct genetic alterations. Here we sought to characterize the pathologic and genetic features of the HER2-positive and HER2-negative components of breast cancers with heterogeneous HER2 gene amplification and to define the repertoire of potential driver genetic alterations in the HER2-negative components of these cases. RESULTS: We separately analyzed the HER2-negative and HER2-positive components of 12 HER2 heterogeneous breast cancers using gene copy number profiling and massively parallel sequencing, and identified potential driver genetic alterations restricted to the HER2-negative cells in each case. In vitro experiments provided functional evidence to suggest that BRF2 and DSN1 overexpression/amplification, and the HER2 I767M mutation may be alterations that compensate for the lack of HER2 amplification in the HER2-negative components of HER2 heterogeneous breast cancers. CONCLUSIONS: Our results indicate that even driver genetic alterations, such as HER2 gene amplification, can be heterogeneously distributed within a cancer, and that the HER2-negative components are likely driven by genetic alterations not present in the HER2-positive components, including BRF2 and DSN1 amplification and HER2 somatic mutations.

Integrative analyses identify modulators of response to neoadjuvant aromatase inhibitors in patients with early breast cancer.

INTRODUCTION: Aromatase inhibitors (AIs) are a vital component of estrogen receptor positive (ER+) breast cancer treatment. De novo and acquired resistance, however, is common. The aims of this study were to relate patterns of copy number aberrations to molecular and proliferative response to AIs, to study differences in the patterns of copy number aberrations between breast cancer samples pre- and post-AI neoadjuvant therapy, and to identify putative biomarkers for resistance to neoadjuvant AI therapy using an integrative analysis approach. METHODS: Samples from 84 patients derived from two neoadjuvant AI therapy trials were subjected to copy number profiling by microarray-based comparative genomic hybridisation (aCGH, n=84), gene expression profiling (n=47), matched pre- and post-AI aCGH (n=19 pairs) and Ki67-based AI-response analysis (n=39). RESULTS: Integrative analysis of these datasets identified a set of nine genes that, when amplified, were associated with a poor response to AIs, and were significantly overexpressed when amplified, including CHKA, LRP5 and SAPS3. Functional validation in vitro, using cell lines with and without amplification of these genes (SUM44, MDA-MB134-VI, T47D and MCF7) and a model of acquired AI-resistance (MCF7-LTED) identified CHKA as a gene that when amplified modulates estrogen receptor (ER)-driven proliferation, ER/estrogen response element (ERE) transactivation, expression of ER-regulated genes and phosphorylation of V-AKT murine thymoma viral oncogene homolog 1 (AKT1). CONCLUSIONS: These data provide a rationale for investigation of the role of CHKA in further models of de novo and acquired resistance to AIs, and provide proof of concept that integrative genomic analyses can identify biologically relevant modulators of AI response.

Integrative genomic and transcriptomic characterization of papillary carcinomas of the breast.

Papillary carcinoma (PC) is a rare type of breast cancer, which comprises three histologic subtypes: encapsulated PC (EPC), solid PC (SPC) and invasive PC (IPC). Microarray-based gene expression and Affymetrix SNP 6.0 gene copy number profiling, and RNA-sequencing revealed that PCs are luminal breast cancers that display transcriptomic profiles distinct from those of grade- and estrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs), and that the papillary histologic pattern is unlikely to be underpinned by a highly recurrent expressed fusion gene or a highly recurrent expressed mutation. Despite displaying similar patterns of gene copy number alterations, significant differences in the transcriptomic profiles of EPCs, SPCs and IPCs were found, and may account for their different histologic features.

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast.

Micropapillary carcinoma (MPC) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations (CNAs) distinct from that of grade- and oestrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray-based comparative genomic hybridization (aCGH) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs. Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC-NSTs, and recurrent mutations affecting mitogen-activated protein kinase family genes and NBPF10. RNA-sequencing analysis identified 17 high-confidence fusion genes, eight of which were validated and two of which were in-frame. No recurrent fusions were identified in an independent series of MPCs and IC-NSTs. Forced expression of in-frame fusion genes (SLC2A1-FAF1 and BCAS4-AURKA) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out-of-frame rearrangements was found in one MPC and in 13% of HER2-positive breast cancers, identified through a re-analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild-type CDK12 in a CDK12-null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities.

Mutation profiling of adenoid cystic carcinomas from multiple anatomical sites identifies mutations in the RAS pathway, but no KIT mutations.

AIMS: The majority of adenoid cystic carcinomas (AdCCs), regardless of anatomical site, harbour the MYB-NFIB fusion gene. The aim of this study was to characterize the repertoire of somatic genetic events affecting known cancer genes in AdCCs. METHODS AND RESULTS: DNA was extracted from 13 microdissected breast AdCCs, and subjected to a mutation survey using the Sequenom OncoCarta Panel v1.0. Genes found to be mutated in any of the breast AdCCs and genes related to the same canonical molecular pathways, as well as KIT, a proto-oncogene whose protein product is expressed in AdCCs, were sequenced in an additional 68 AdCCs from various anatomical sites by Sanger sequencing. Using the Sequenom MassARRAY platform and Sanger sequencing, mutations in BRAF and HRAS were identified in three and one cases, respectively (breast, and head and neck). KIT, which has previously been reported to be mutated in AdCCs, was also investigated, but no mutations were identified. CONCLUSIONS: Our results demonstrate that mutations in genes pertaining to the canonical RAS pathway are found in a minority of AdCCs, and that activating KIT mutations are either absent or remarkably rare in these cancers, and unlikely to constitute a driver and therapeutic target for patients with AdCC.

The 11q13-q14 amplicon: clinicopathological correlations and potential drivers.

Amplification at 11q13-q14 is a common event in cancers from multiple anatomical sites. This complex amplicon has multiple cores and several genes have been put forward as potential "drivers." In this review, based on the technical advancements of the last decade, which resulted in methods allowing for a deeper genomic and functional genomic characterization of amplicons and their drivers, we discuss the current definitions of amplicons and driver genes, the clinical and biological significance of the 11q13-q14 amplicon in distinct types of human cancer, its coamplification partners, and the roles of various genes located within the amplicon as potential "drivers." Finally, we appraise the available data for novel therapies targeting genes mapping to this amplicon.

Functional characterization of the 19q12 amplicon in grade III breast cancers.

INTRODUCTION: The 19q12 locus is amplified in a subgroup of oestrogen receptor (ER)-negative grade III breast cancers. This amplicon comprises nine genes, including cyclin E1 (CCNE1), which has been proposed as its 'driver'. The aim of this study was to identify the genes within the 19q12 amplicon whose expression is required for the survival of cancer cells harbouring their amplification. METHODS: We investigated the presence of 19q12 amplification in a series of 313 frozen primary breast cancers and 56 breast cancer cell lines using microarray comparative genomic hybridisation (aCGH). The nine genes mapping to the smallest region of amplification on 19q12 were silenced using RNA interference in phenotypically matched breast cancer cell lines with (MDA-MB-157 and HCC1569) and without (Hs578T, MCF7, MDA-MB-231, ZR75.1, JIMT1 and BT474) amplification of this locus. Genes whose silencing was selectively lethal in amplified cells were taken forward for further validation. The effects of cyclin-dependent kinase 2 (CDK2) silencing and chemical inhibition were tested in cancer cells with and without CCNE1 amplification. RESULTS: 19q12 amplification was identified in 7.8% of ER-negative grade III breast cancer. Of the nine genes mapping to this amplicon, UQCRFS1, POP4, PLEKHF1, C19ORF12, CCNE1 and C19ORF2 were significantly over-expressed when amplified in primary breast cancers and/or breast cancer cell lines. Silencing of POP4, PLEKHF1, CCNE1 and TSZH3 selectively reduced cell viability in cancer cells harbouring their amplification. Cancer cells with CCNE1 amplification were shown to be dependent on CDK2 expression and kinase activity for their survival. CONCLUSIONS: The 19q12 amplicon may harbour more than a single 'driver', given that expression of POP4, PLEKHF1, CCNE1 and TSZH3 is required for the survival of cancer cells displaying their amplification. The observation that cancer cells harbouring CCNE1 gene amplification are sensitive to CDK2 inhibitors provides a rationale for the testing of these chemical inhibitors in a subgroup of patients with ER-negative grade III breast cancers.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers.

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Genomic and mutational profiling of ductal carcinomas in situ and matched adjacent invasive breast cancers reveals intra-tumour genetic heterogeneity and clonal selection.

The mechanisms underlying the progression from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) of the breast are yet to be fully elucidated. Several hypotheses have been put forward to explain the progression from DCIS to IDC, including the selection of a subpopulation of cancer cells with specific genetic aberrations, and the acquisition of new genetic aberrations or non-genetic mechanisms mediated by the tumour microenvironment. To determine whether synchronously diagnosed ipsilateral DCI and IDCs have modal populations with distinct repertoires of gene copy number aberrations and mutations in common oncogenes, matched frozen samples of DCIS and IDC were retrieved from 13 patients and subjected to microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY (Oncocarta v 1.0 panel). Fluorescence in situ hybridization and Sanger sequencing were employed to validate the aCGH and Sequenom findings, respectively. Although the genomic profiles of matched DCI and IDCs were similar, in three of 13 matched pairs amplification of distinct loci (ie 1q41, 2q24.2, 6q22.31, 7q11.21, 8q21.2 and 9p13.3) was either restricted to, or more prevalent in, the modal population of cancer cells of one of the components. Sequenom MassARRAY identified PIK3CA mutations restricted to the DCIS component in two cases, and in a third case the frequency of the PIK3CA mutant allele reduced from 49% in the DCIS to 25% in the IDC component. Despite the genomic similarities between synchronous DCIS and IDC, our data provide strong circumstantial evidence to suggest that in some cases the progression from DCIS to IDC is driven by the selection of non-modal clones that harbour a specific repertoire of genetic aberrations.

Immunophenotypic and genomic characterization of papillary carcinomas of the breast.

Papillary carcinomas are a special histological type of breast cancer and have a relatively good outcome. We characterized the genomic and phenotypic characteristics of papillary carcinomas to determine whether they would constitute an entity distinct from grade- and oestrogen receptor (ER)-matched invasive ductal carcinomas of no special type (IDC-NSTs). The phenotype of 63 papillary carcinomas of the breast and grade- and ER-matched IDC-NSTs was determined by immunohistochemistry. DNA of sufficient quality was extracted from 49 microdissected papillary carcinomas and 49 microdissected grade- and ER-matched IDC-NSTs. These samples were subjected to high-resolution microarray-based comparative genomic hybridization (aCGH) and Sequenom MassARRAY sequencing analysis of 19 known oncogenes. Papillary carcinomas were predominantly of low histological grade, expressed immunohistochemical markers consistent with a luminal phenotype, and a lower rate of lymph node metastasis and p53 expression than grade- and ER-matched IDC-NSTs. Papillary carcinomas displayed less genomic aberrations than grade- and ER-matched IDC-NSTs; however, the patterns of gene copy number aberrations found in papillary carcinomas were similar to those of ER- and grade-matched IDC-NSTs, including 16q losses. Furthermore, PIK3CA mutations were found in 43% and 29% of papillary carcinomas and grade- and ER-matched IDC-NSTs, respectively. The genomic profiles of encapsulated, solid and invasive papillary carcinomas, the three morphological subtypes, were remarkably similar. Our results demonstrate that papillary carcinomas are a homogeneous special histological type of breast cancer. The similarities in the genomic profiles of papillary carcinomas and grade- and ER-matched IDC-NSTs suggest that papillary carcinomas may be best positioned as part of the spectrum of ER-positive breast cancers, rather than as a distinct entity. Furthermore, the good prognosis of papillary carcinomas may stem from the low rates of lymph node metastasis and p53 expression, low number of gene copy number aberrations and high prevalence of PIK3CA mutations.

High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform.

Fusion genes have pivotal roles in the development and progression of human cancer and offer potential for rational drug design. Massively parallel sequencing has identified a panoply of in-frame expressed fusion genes, but early reports suggest that the majority of these are present at very low prevalence or are private events. Conventional methods for the identification of recurrent expressed fusion genes in large cohorts of cancers (eg fluorescence in situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR)) are time consuming and prone to artifacts. Here, we describe a novel high-throughput strategy for the detection of recurrent fusion genes in cancer based on the Sequenom MassARRAY platform. Fusion genes were initially identified by massively parallel sequencing of breast cancer cell lines. For each fusion gene, two Sequenom probes were designed. Primary human breast cancers and cancer cell lines were interrogated for 10 fusion genes. Sensitivity, specificity, and predictive values of the MassARRAY method were then determined using FISH and qRT-PCR as the 'gold standard.' By combining two probes per fusion gene, the negative and positive predictive values were 100 and 71.4%, respectively. All fusion genes identified by massively parallel sequencing were accurately detected. No recurrent fusion genes were found. The MassARRAY-based approach described here may, therefore, be employed as a high-throughput screening tool for known fusion genes in human cancer. In keeping with other highly sensitive assays, further refinement of this technique is necessary to reduce the number of false-positive results.

Functional characterization of EMSY gene amplification in human cancers.

The 11q13-q14 locus is frequently amplified in human cancers, with a complex structure harbouring multiple potential oncogenic drivers. The EMSY gene has been proposed as a driver of the third core of the 11q13-q14 amplicon. This gene encodes a protein reported to be a BRCA2-binding partner, which when over-expressed would lead to impairment of BRCA2 functions and could constitute a mechanism for BRCA2 inactivation in non-hereditary breast and ovarian cancers. We hypothesized that if EMSY amplification abrogates BRCA2 functions, cells harbouring this aberration would be unable to elicit competent homologous recombination DNA repair and, therefore, may have increased sensitivity to genotoxic therapies and potent PARP inhibitors. Microarray-based comparative genomic hybridization of cell lines from distinct tumour sites, including breast, ovary, pancreas, oesophagus, lung and the oral cavity, led to the identification of 10 cell lines with EMSY amplification and 18 without. EMSY amplification was shown to correlate with EMSY mRNA levels, although not all cell lines harbouring EMSY amplification displayed EMSY mRNA or protein over-expression. RNA interference-mediated silencing of EMSY did not lead to a reduction in cell viability in tumour models harbouring EMSY amplification. Cell lines with and without EMSY amplification displayed a similar ability to elicit RAD51 foci in response to DNA damaging agents, and similar sensitivity to cisplatin and olaparib. Taken together, this suggests that EMSY is unlikely to be a driver of the 11q13-q14 amplicon and does not have a dominant role in modulating the response to agents targeting cells with defective homologous recombination.

Synthetic lethality of PARP inhibition in cancers lacking BRCA1 and BRCA2 mutations.

Utilizing the concept of synthetic lethality has provided new opportunities for the development of targeted therapies, by allowing the targeting of loss of function genetic aberrations. In cancer cells with BRCA1 or BRCA2 loss of function, which harbor deficiency of DNA repair by homologous recombination, inhibition of PARP1 enzymatic activity leads to an accumulation of single strand breaks that are converted to double strand breaks but cannot be repaired by homologous recombination. Inhibition of PARP has therefore been advanced as a novel targeted therapy for cancers harboring BRCA1/2 mutations. Preclinical and preliminary clinical evidence, however, suggests a potentially broader scope for PARP inhibitors. Loss of function of various proteins involved in double strand break repair other than BRCA1/2 has been suggested to be synthetically lethal with PARP inhibition. Inactivation of these genes has been reported in a subset of human cancers and might therefore constitute predictive biomarkers for PARP inhibition. Here we discuss the evidence that the clinical use of PARP inhibition may be broader than targeting of cancers in BRCA1/2 germ-line mutation carriers.

Thoracobilia: a rare complication of gallstone disease.

Thoracobilia and pleurobiliary fistulae secondary to obstructed choledocholithiasis are rare complications of gallstone disease. We present the case of a 64-year-old man with gallstone pancreatitis who developed a pleurobiliary fistula secondary to an obstructing ductal calculus, and was successfully managed non-operatively.